The present invention provides novel coding sequences for use in plants. The coding sequences encode a chimeric insecticidal protein toxic to a wide range of lepidopteran species crop pests.
Commercial formulations of naturally occurring B. thuringiensis isolates have long been used for the biological control of agricultural insect pests. Bt spores and crystals obtained from fermentation of Bacillus thuringiensis species are concentrated and formulated for foliar application according to conventional agricultural practices.
Members of the family of Cry1 crystal proteins are known to exhibit bioactivity against lepidopteran insect larvae and are useful as agents for controlling lepidopteran insect pests. The precursor form of Cry1 δ-endotoxins consist of two approximately equal sized segments. The carboxy-terminal portion of the precursor protein, or pro-toxin segment, stabilizes crystal formation and exhibits no insecticidal activity. The amino-terminal half of the precursor protein comprises the toxin segment of the Cry1 protein and, based on alignment of conserved or substantially conserved sequences within Cry1 family members, can be further sub-divided into three structural domains. These three sub-domains are based on a three dimensional crystallographic structural model of a Cry1A δ-endotoxin in which the three sub-domains were referred to as Domain I, Domain II, and Domain III, respectively as measured from the amino terminus of the protein toxin segment. Domain I comprises about the first third of the active toxin segment and has been shown to be essential for channel formation (Thompson et al., 1995). Domains II and III respectively comprise about the central and carboxy-terminal segments of the active toxin portion. Domains II and III have both been implicated in receptor binding and insect species specificity, depending on the insect and δ-endotoxin being examined (Thompson et al., 1995).
The likelihood of arbitrarily creating a chimeric protein with enhanced properties from the reassortment of the domain structures of the numerous native insecticidal crystal proteins known in the art is remote. This is a result of the complex nature of protein structure, folding, oligomerization, and activation including correct proteolytic processing of the chimeric precursor, if expressed in such form, to release an insecticidal toxin segment. Only by careful selection of specific target regions within each parental protein for inclusion into a chimeric structure can functional insecticidal toxins be constructed that exhibit improved insecticidal activity in comparison to the parental proteins from which the chimeras are derived. Experience has shown that reassembly of the toxin domains, i.e., assembly of a chimeric toxin consisting of domain I, II, and III of any two or more toxins that are different from each other, results in the construction of a protein that exhibits faulty crystal formation and/or the complete lack of any detectable insecticidal activity directed to a preferred target insect pest species. In some instances, a chimeric toxin will exhibit good crystal formation properties, yet exhibit no detectable insecticidal activity. Only by trial and error are effective insecticidal chimeras formulated, and even then, the skilled artisan is not certain to end up with a chimera that exhibits insecticidal activity that is equivalent to or improved in comparison to any single parental toxin protein from which the constituents of the chimera may have been derived.
The literature reports examples of the construction or assembly of chimeric proteins from two or more Bt insecticidal crystal protein precursors, yet not all exhibited insecticidal or crystal forming properties that were equivalent to or improved in comparison to the precursor proteins from which the chimeras were derived. (Bosch et al. (WO95/06730); Thompson et al. (WO95/30753); Thompson et al. (WO95/30752); Malvar et al. (WO98/22595); Gilroy et al. (U.S. Pat. No. 5,128,130); Gilroy (U.S. Pat. No. 5,055,294); Lee et al. (1992) Gene 267:3115-3121; Honee et al. (1991) Mol. Microbiol. 5:2799-2806; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930; Perlak et al. (1990) Bio/Technol. 8:939-9943; Perlak et al (1993) Plant Mol. Biol. 22:313-321).
Expression of B. thuringiensis δ-endotoxins in transgenic corn plants has proven to be an effective means of controlling agriculturally important insect pests (Perlak et al. 1990; 1993). Transgenic crops expressing B. thuringiensis δ-endotoxins enable growers to significantly reduce the time and costs associated with applications of topically applied chemical insecticides. Use of transgenes encoding B. thuringiensis δ-endotoxins is particularly advantageous. Crop plants expressing B. thuringiensis δ-endotoxins in areas under heavy insect pressure exhibit improved yields that are better than otherwise similar non-transgenic commercial plant varieties. However, it is anticipated that insects may evolve resistance to B. thuringiensis δ-endotoxins expressed in transgenic plants. Such resistance, should it become widespread, would clearly limit the commercial value of germplasm containing genes encoding such B. thuringiensis δ-endotoxins. One possible way of increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests would be to ensure that transgenic crops express high levels of B. thuringiensis δ-endotoxins (McGaughey and Whalon 1993; Roush 1994). In addition, having a repository of insecticidal genes that are effective against groups of insect pests and which manifest their effects through different modes of action can safeguard against any development of resistance. Expression in a plant of two or more insecticidal compositions toxic to the same insect species, each insecticide being expressed at levels high enough to effectively delay the onset of resistance, would be another way to achieve control of the development of resistance. Examples of such insecticides useful in such combinations include but are not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, deallergenized and de-glycosylated patatin proteins and/or permuteins, plant lectins, and the like. Achieving co-expression of multiple insecticidally active proteins in the same plant, and/or high expression levels of those insecticidal proteins without causing undesirable plant morphological effects has been elusive.
Only a handful of the more than two-hundred and fifty individual insecticidal proteins that have been identified from Bacillus thuringiensis species have been tested for expression in plants. Several Cry1's, Cry3's, Cry2Aa and Cry2Ab, binary toxins Cry33/34 and Cry23/37, and a Cry9 have been successfully expressed in plants. Cry1 proteins represent the largest class of proteins that have been expressed in plants, but none have been expressed at high levels. It was necessary to target the Cry2Ab to the chloroplast to avoid undesirable phytotoxic effects. The majority of acres planted in recombinant plants express Cry1A proteins. The likelihood of the onset of resistance to Cry1A proteins by targeted insect pest species is substantially higher than it would be if a resistance management allele was also expressed along with the cry1 allele, or if the cry1 allele was expressed at high levels. Therefore it is desirable that alternative toxin genes be developed for expression in plants as supplements and replacements for those being used presently in the first and second generations of transgenic insect resistant plants.